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Obtaining DNA nanostructures with potential applications in drug discovery, diagnostics, and electronics in a simple and affordable way represents one of the hottest topics in nanotechnological and medical sciences. Herein, we report a novel strategy to obtain structurally homogeneous DNA G‐wire nanostructures of known length, starting from the short unmodified G‐rich oligonucleotide d(5′‐CGGT‐3′–3′‐GGC‐5′) (1) incorporating a 3’–3′ inversion of polarity site. The reported approach allowed us to obtain long G‐wire assemblies through 5′–5′ π–π stacking interactions in between the tetramolecular G‐quadruplex building blocks that form when 1 is annealed in the presence of potassium ions. Our results expand the repertoire of synthetic methodologies to obtain new tailored DNA G‐wire nanostructures.

Oligonucleotides and oligonucleotide analogues are extremely versatile molecules from a biotechnological point of view, since they could be used as scaffolds for the development of new drugs in therapeutics, but also for the development of new materials in nanotechnology. The present thesis is included in this context and it is aimed at the discovery of all potential applications of these molecules. In particular, my work is mainly focused on: ✓ The synthesis and characterization of a novel G-rich ODN sequence for the development of long G-wires; ✓ The synthesis and characterization of PNA sequences to be used in a novel approach for the treatment of Cystic Fibrosis; ✓ The synthesis and characterization of a short sequence of PNA conjugated to a novel bifunctional linker aimed at the obtainment of PNA-based quadruplex.

A general synthesis of C6-pyridylpurine nucleosides is described. The reported synthetic procedure exploits the regioselective addition of pyridinyl Grignard reagents, obtained by bromine/magnesium exchange between mono- or dihalopyridines and iPrMgCl, to the C6–N1–O– moiety of nebularine N1-oxide. The regioselective transmetallation of unsymmetrical dihalopyridines with iPrMgCl allowed C6-halopyridylpurine nucleosides to be obtained through the addition of halopyridinyl Grignard reagents. The presence of a halogenated pyridine ring in these nucleosides allows for further useful synthetic transformations.

Cyclic N1-pentylinosine monophosphate (cpIMP), a novel simplified inosine derivative of cyclic ADP-ribose (cADPR) in which the N1-pentyl chain and the monophosphate group replace the northern ribose and the pyrophosphate moieties, respectively, was synthesized. The role played by the position of the phosphate group in the key cyclization step, which consists in the formation of a phosphodiester bond, was thoroughly investigated. We have also examined the influence of the phosphate bridge on the ability of cpIMP to mobilize Ca2+ in PC12 neuronal cells in comparison with the pyrophosphate bridge present in the cyclic N1-pentylinosine diphosphate analogue (cpIDP) previously synthesized in our laboratories. The preliminary biological tests indicated that cpIMP and cpIDP induce a rapid increase of intracellular Ca2+ concentration in PC12 neuronal cells.

Background G-quadruplex DNA is involved in many physiological and pathological processes. Both clinical and experimental studies on DNA G-quadruplexes are slowed down by their enzymatic instability. In this frame, more stable chemically modified analogs are needed. Methods The bis-end-linked-(gggt) 2 PNA molecule (BEL-PNA) was synthesized using in solution and solid phase synthetic approaches. Quadruplex formation was assessed by circular dichroism (CD) and surface enhanced Raman scattering (SERS). Results An unprecedented bimolecular PNA homo quadruplex is here reported. To achieve this goal, we developed a bifunctional linker that once functionalized with gggt PNA strands and annealed in K + buffer allowed the obtainment of a PNA homo quadruplex. The identification of the strong SERS band at ~ 1481 cm − 1 , attributable to vibrations involving the quadruplex diagnostic Hoogsteen type hydrogen bonds, confirmed the formation of the PNA homo quadruplex. Conclusions By tethering two G-rich PNA strands to the two ends of a suitable bifunctional linker it is possible to obtain bimolecular PNA homo quadruplexes after annealing in K + -containing buffers. The formation of such CD-unfriendly complexes can be monitored, even at low concentrations, by using the SERS technique. General significance Given the importance of DNA G-quadruplexes in medicine and nanotechnology, the obtainment of G-quadruplex analogs provided with enhanced enzymatic stability, and their monitoring by highly sensitive label-free techniques are of the highest importance. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.